Crystallization and preliminary X-ray diffraction analysis of restriction endonuclease EcoRII

Abstract

Crystals of the restriction endonuclease EcoRII have been obtained by the vapor-diffusion technique in the presence of ammonium sulfate or polyethylene glycol. The best crystals were grown with ammonium sulfate as a precipitant. Crystals with dimensions of up to 0.6 Â 0.6 Â 0.6 mm have been observed. The crystals diffract to about 4.0 A Ê resolution at a cryo-temperature of 100 K using a rotatinganode X-ray source and a Rigaku R-AXIS IV imaging-plate detector. The space group has been determined to be either I23 or I2 1 3, with unit-cell parameters a = b = c = 160.3 A Ê , = = = 90 . The crystal asymmetric unit contains two protein molecules, and self-rotation function analysis shows a pseudo-twofold symmetry relating the two monomers. Attempts to improve the resolution of crystal diffraction and to search for heavy-atom derivatives are under way.

A new crystal form of restriction endonuclease EcoRII that diffracts to 2.8 Å resolution

“…The other two peaks with the second highest peak height of about 75 at (i) 9 = 30 and 2 = 90 and (ii) 9 = 120 and 2 = 90 are noncrystallographic twofold axes relating the two monomers in the asymmetric unit. The positions of the two pseudo-twofold axes in relation to the crystallographic twofold axis in the rotation map imply that the crystal may possess pseudo-222 symmetry, while the relative peak heights indicate that the crystal (Karpova et al, 1999). They diffracted poorly to 4 A Ê resolution at best, while the monoclinic form crystals diffracted to much higher resolution (2.8 A Ê ) on a synchrotron source.…”

“…The cubic form crystals were obtained after a long period of extensive screening and optimization (Karpova et al, 1999). However, these crystals diffracted poorly to less than 4 A Ê resolution on a rotating-anode X-ray source.…”

“…EcoRII was ®rst crystallized in the cubic space group I23 or I2 1 3 (Karpova et al, 1999). However, the cubic crystal form diffracted only to 4 A Ê resolution at best.…”